D. Intracellular Ca2+ Transients in Neurons and Glia
VI. Conclusions and Future Prospects
The development of novel fluorescent probes of cellular structure and physiology (Mason, 1993; Tsien and Waggoner, 1995) has had a profound impact on studies of brain structure and function at the network, cellular, and subcellular levels.
Coupled with the technical advances in high-resolution optical imaging, fluorescent markers provide a valuable set of
Figure 18Patterns of picrotoxin-induced epileptiform activity in hippocampal slices detected by fast-scanning confocal imaging of intracellular calcium. The high time resolution was achieved using a confocal microscope (Noran Odyssey) that collects full-field images at video rate (30 Hz). (A) A single scan (nonaveraged) image showing the relatively low spatial resolution of fluo-3-labeled pyramidal neurons when imaged at high time resolution (compare with slow-scanned images in Figs. 16 and 17A). The slice is oriented such that the pyramidal cell body layer (SP) runs in a horizontal band across the middle of the field. Numbered boxes indicate the location of some of the pyramidal cell bodies, which were more clearly evident in averaged images.
SR, stratum radiatum. SO, stratum oriens. Scale bar, 20 µm. (B) Plots of fluorescence intensity over time for the five boxed regions, corresponding to five pyramidal cell bodies, in A. Note how the high time resolution of the fast-scanning imaging reveals the repetitive, synchronized Ca2+spikes in the neurons.
(C) Plots of fluorescence intensity for a neuron (upper trace) and for four other nearby cells (not shown) that are most likely glia. Note the various patterns and slower time courses of Ca2+transients in the nonneuronal cells that are resolved with the fast-scanning imaging.
tools for mapping the functional organization of the brain.
The wide variety of fluorescent membrane dyes now avail- able with varying spectral properties permits simultaneous labeling and discrimination of different populations of cells.
This will continue to be useful to hodologists interested in more precisely identifying the organization and interrelation- ship of neural projections in brain tissues.
One area of great potential growth is in the development of new structural and functional probes that would be useful for identifying specific populations of neurons or for labeling functional synaptic contacts in live brain tissues. Ideal probes will permit functional analysis at high spatial and temporal resolution. The widespread use of GFP and similar proteins indicates that genetically engineered fluorescent probes should continue to play an important role in elucidating neural structure and function (see Yuste et al., 1999a). New probes that are likely to participate significantly in this endeavor include calcium-sensitive (Miyawaki et al., 1997), pH-sensitive (Miesenbock et al., 1998), and voltage-sensi- tive (Siegel and Isacoff, 1997, 1999; Sakai et al., 2001) fluorescent proteins, especially those that are targeted to synapses (Sankaranarayanan et al., 2000). It seems likely that genetic probes whose expression can be restricted to certain brain regions or cell types will contribute signifi- cantly toward mapping the functional anatomy of the brain (Spergel et al., 2001). Already, there are several transgenic mouse models that express reporters such as GFP in
restricted subsets of neurons (van den Pol and Ghosh, 1998;
Oliva et al., 2000) or glia (Zhuo et al., 1997).
Given the advancements in vital fluorescent probes and sensitive imaging techniques, it is now possible to map the 3D structure of single neurons and glial cells in live brain slices over a period of many hours. The ability to collect 2D and 3D image data sets from live neural tissue slices at high spatial resolution, over long periods of time and at relatively short time intervals, is revealing new information on the dynamics of neural structure in brain tissues. The time-resolved 3D imaging methods described here capture more of the dynamic events occurring within tissue and also provide the researcher with assurance that observed structural changes are not due to movement in and out of a focal plane. We can expect these vital fluorescence labeling and imaging methods to be applied more widely to studies of structural and functional neural organization in a variety of brain regions, especially with the use of genetic probes that can be targeted to specific brain regions and cell types. Moreover, it is anticipated that cell- type-specific probes will enable new strategies for investigat- ing structural and functional relationships of neurons and glia.
Much can be done with the existing optical imaging tech- nology. Nevertheless, future developments will undoubtedly continue to address constraints on high-resolution imaging deep (>50 µm) within tissues. Improvements are already being realized by using water-immersion lenses (to reduce spherical aberration) and longer wavelength dyes (to reduce Figure 19 Glial cell Ca2+transients in area CA3 of an organotypic hippocampal brain slice. This
is a composite image showing Ca2+activity at three different time points (encoded blue–green–red, respectively) collected at 7-s intervals. The layer of pyramidal cell bodies (SP) runs diagonal from upper left to lower right. Cell bodies and fine cellular processes of these nonneuronal cells, most prob- ably astrocytes, are spontaneously active. Astrocyte activity is most prominent in the layers (SR and SP) adjacent to the pyramidal cell body layer, where there is a high density of excitatory synaptic con- tacts onto pyramidal cell dendrites. Intercellular waves of Ca2+activity have been shown to propagate over long distances through such organized glial networks (see text). Scale bar, 50 µm.
light scatter by the tissue and minimize phototoxic effects).
Newer optical techniques such as multiphoton imaging (Denk et al., 1990, 1994) are being used more widely and are making significant contributions to mapping neural structure and function. Multiphoton imaging provides intrinsic three- dimensional resolution (Williams et al., 1994) while confining fluorescence excitation to a single narrow focal plane, thus reducing the risk of photodynamic damage. With regard to imaging cellular and subcellular structure, multi- photon excitation yields a substantial improvement over con- ventional confocal for imaging fluorescently labeled cells at deeper (100–500 µm) levels in live tissues (Mainen et al., 1999; Majewska et al., 2000).
Another very exciting optical technique that should greatly facilitate the mapping of functional neural organization in brain slices is based on laser photostimulation (Farber and Grinvald, 1983). A modification of this technique (Callaway and Katz, 1993; Katz and Dalva, 1994) employs a scanned laser beam to focally release a caged (photoactivatable) form of the excitatory neurotransmitter, glutamate, thus stimulating nearby neurons. The development of new probes that can be uncaged with multiphoton excitation (Augustine, 2001;
Matsuzaki et al., 2001) should enable higher resolution studies in more intact tissues. In conjunction with electro- physiological recordings or physiological imaging, these methods should continue to provide opportunities for high- resolution mapping of neural circuits in excised brain slices.
At present, most of the work at the cellular and subcellu- lar levels of resolution is being done in excised tissue slices.
However, future work will continue to probe more of the outstanding questions in intact, functioning preparations (e.g., Dirnagl et al., 1991; Them, 1993; Svoboda et al., 1999; Lendvai et al., 2000; Ilyin et al., 2001). At any rate, it is clear that neuroscientists have a repertoire of powerful optical tools for dissecting and mapping the functional organization of brain tissues.
Acknowledgments
Some of the work described herein was performed by the author in the lab- oratory of Dr. Stephen J. Smith (Stanford University). More recent work in the author’s laboratory at the University of Iowa was supported by grants from the NIH (NS37159) and Whitehall Foundation (98-6).
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